Mechanical stability of αT-catenin and its activation by force for vinculin binding

alpha T (Testes)-catenin, a critical factor regulating cell-cell adhesion in the heart, directly couples the cadherin-catenin complex to the actin cytoskeleton at the intercalated disk (ICD), a unique cell-cell junction that couples cardiomyocytes. Loss of alpha T-catenin in mice reduces plakophilin2 and connexin 43 recruitment to the ICD. Since alpha T-catenin is subjected to mechanical stretch during actomyosin contraction in cardiomyocytes, its activity could be regulated by mechanical force. To provide insight in how force regulates alpha T-catenin function, we investigated the mechanical stability of the putative, force-sensing middle (M) domain of alpha T-catenin and determined how force impacts vinculin binding to alpha T-catenin. We show that 1) physiological levels of force, <15 pN, are sufficient to unfold the three M domains; 2) the M1 domain that harbors the vinculin-binding site is unfolded at similar to 6 pN; and 3) unfolding of the M1 domain is necessary for high-affinity vinculin binding. In addition, we quantified the binding kinetics and affinity of vinculin to the mechanically exposed binding site in M1 and observed that alpha T-catenin binds vinculin with low nanomolar affinity. These results provide important new insights into the mechanosensing properties of alpha T-catenin and how alpha T-catenin regulates cell-cell adhesion at the cardiomyocyte ICD.